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De-SUMOylation of FOXC2 by SENP3 promotes the epithelial-mesenchymal transition in gastric cancer cells.

Ren YH, Liu KJ, Wang M, Yu YN, Yang K, Chen Q, Yu B, Wang W, Li QW, Wang J, Hou ZY, Fang JY, Yeh ET, Yang J, Yi J - Oncotarget (2014)

Bottom Line: The impact of cellular oxidative stress in promoting the epithelial-mesenchymal transition (EMT) has been noticed.Meanwhile N-cadherin is verified as a target gene of FOXC2, which is transcriptionally activated by a SUMO-less FOXC2.Additionally, reactive oxygen species-induced de-SUMOylation of FOXC2 can be blocked by silencing endogenous SENP3.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Cell Biology, Shanghai Key Laboratory of Tumor Microenvironment and Inflammation, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China. Institute of Neuroscience, Wenzhou Medical University, School of Medicine, Zhejiang, China. These authors contribute equally to the work.

ABSTRACT
The impact of cellular oxidative stress in promoting the epithelial-mesenchymal transition (EMT) has been noticed. Our previous study shows that SENP3, a redox-sensitive SUMO2/3-specific protease, accumulates in a variety of cancers, but whether SENP3 and SUMOylation involve in the regulation of EMT is unclear. The present study uncovers a novel role of SENP3 in promoting the EMT process in gastric cancer via regulating an EMT-inducing transcription factor, forkhead box C2 (FOXC2). We demonstrate that the expression of mesenchymal marker genes and cell migration ability are enhanced in SENP3-overexpressing gastric cancer cells and attenuated in SENP3-knockdown cells. A nude mouse model and a set of patient's specimens suggest the correlation between SENP3 and gastric cancer metastasis. Biochemical assays identify FOXC2 as a substrate of SENP3. Meanwhile N-cadherin is verified as a target gene of FOXC2, which is transcriptionally activated by a SUMO-less FOXC2. Additionally, reactive oxygen species-induced de-SUMOylation of FOXC2 can be blocked by silencing endogenous SENP3. In conclusion, SENP3, which is increased in gastric cancer cells, potentiates the transcriptional activity of FOXC2 through de-SUMOylation, in favor of the induction of specific mesenchymal gene expression in gastric cancer metastasis.

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SENP3 upregulates the transcription of N-cadherin through de-SUMOlyation of FOXC2(A) Luciferase reporter assay in HEK293T cells co-transfected with the vector/N-cadherin promoter reporter and different doses of FOXC2. Relative luciferase activity (RLA) was shown as mean±SEM of three independent experiments. (B) The constructs of a series of N-cadherin promoter reporter with various truncations (left) and luciferase reporter assay (right) in HEK293T cells that were co-transfected with the vector/the reporter truncates and FOXC2. (C) Flag ChIP assay using the antibody against Flag and the primer of the N-cadherin promoter region −428/−209 in HEK293T cells that were transfected with the Flag-tagged vector (F-vector) / Flag-tagged wild-type FOXC2 (F-FOXC2). (D, E) Luciferase reporter assay (left) and Flag ChIP (right) in HEK293T cells that were transfected with F-FOXC2 /SUMO-less mutant of FOXC2 (F-FOXC2K214R) (D), or together with wild-type/dominant negative mutant of SENP3 (E). Both assays were repeated three times and the results were shown as mean±SEM. **: P < 0.01; ***: P<0.001. (F) The N-cadherin protein levels in the stable cell lines SGC7901-MOCK and SGC7901-SENP3 that were transiently transfected with FOXC2. (G) The SENP3 and N-cadherin protein levels in the representative gastric cancer tissue lysates.
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Figure 5: SENP3 upregulates the transcription of N-cadherin through de-SUMOlyation of FOXC2(A) Luciferase reporter assay in HEK293T cells co-transfected with the vector/N-cadherin promoter reporter and different doses of FOXC2. Relative luciferase activity (RLA) was shown as mean±SEM of three independent experiments. (B) The constructs of a series of N-cadherin promoter reporter with various truncations (left) and luciferase reporter assay (right) in HEK293T cells that were co-transfected with the vector/the reporter truncates and FOXC2. (C) Flag ChIP assay using the antibody against Flag and the primer of the N-cadherin promoter region −428/−209 in HEK293T cells that were transfected with the Flag-tagged vector (F-vector) / Flag-tagged wild-type FOXC2 (F-FOXC2). (D, E) Luciferase reporter assay (left) and Flag ChIP (right) in HEK293T cells that were transfected with F-FOXC2 /SUMO-less mutant of FOXC2 (F-FOXC2K214R) (D), or together with wild-type/dominant negative mutant of SENP3 (E). Both assays were repeated three times and the results were shown as mean±SEM. **: P < 0.01; ***: P<0.001. (F) The N-cadherin protein levels in the stable cell lines SGC7901-MOCK and SGC7901-SENP3 that were transiently transfected with FOXC2. (G) The SENP3 and N-cadherin protein levels in the representative gastric cancer tissue lysates.

Mentions: Although FOXC2 serves as an EMT-inducing TF, its target genes for mesenchymal induction remain unknown. We noticed that the expressions of fibronectin and N-cadherin were consistently regulated by SENP3 in various gain- or loss-of-function settings. To clarify the functions of FOXC2 in transcriptional regulation of these two genes, we constructed the luciferase reporters fused to the promoter regions of the human fibronectin and N-cadherin genes. The relative luciferase activity (RLA) of the reporter for N-cadherin was increased in a FOXC2 dose-dependent manner (Fig. 5A), indicating that FOXC2 might be the transcription factor of N-cadherin, and that the reporter, namely N-cad luc reporter, was usable. In contrast, the RLA of the reporter for fibronectin was not changed by FOXC2 overexpression (data not showed), suggesting that fibronectin was not regulated by FOXC2, despite that it could be upregulated by SENP3. N-cadherin was then selected as a representative EMT marker gene potentially regulated by SENP3 through FOXC2. To identify the FOXC2-binding elements, we measured the reporter RLA in the presence of FOXC2 based on a series of truncates of this N-cad luc reporter. The results suggested that the sequence between −428 and −209 bp on the N-cadherin promoter contained the essential elements regulated by FOXC2 (Fig. 5B). A ChIP assay was further performed, and the results verified the binding of FOXC2 at the −428 ~ −209 region (Fig. 5C).


De-SUMOylation of FOXC2 by SENP3 promotes the epithelial-mesenchymal transition in gastric cancer cells.

Ren YH, Liu KJ, Wang M, Yu YN, Yang K, Chen Q, Yu B, Wang W, Li QW, Wang J, Hou ZY, Fang JY, Yeh ET, Yang J, Yi J - Oncotarget (2014)

SENP3 upregulates the transcription of N-cadherin through de-SUMOlyation of FOXC2(A) Luciferase reporter assay in HEK293T cells co-transfected with the vector/N-cadherin promoter reporter and different doses of FOXC2. Relative luciferase activity (RLA) was shown as mean±SEM of three independent experiments. (B) The constructs of a series of N-cadherin promoter reporter with various truncations (left) and luciferase reporter assay (right) in HEK293T cells that were co-transfected with the vector/the reporter truncates and FOXC2. (C) Flag ChIP assay using the antibody against Flag and the primer of the N-cadherin promoter region −428/−209 in HEK293T cells that were transfected with the Flag-tagged vector (F-vector) / Flag-tagged wild-type FOXC2 (F-FOXC2). (D, E) Luciferase reporter assay (left) and Flag ChIP (right) in HEK293T cells that were transfected with F-FOXC2 /SUMO-less mutant of FOXC2 (F-FOXC2K214R) (D), or together with wild-type/dominant negative mutant of SENP3 (E). Both assays were repeated three times and the results were shown as mean±SEM. **: P < 0.01; ***: P<0.001. (F) The N-cadherin protein levels in the stable cell lines SGC7901-MOCK and SGC7901-SENP3 that were transiently transfected with FOXC2. (G) The SENP3 and N-cadherin protein levels in the representative gastric cancer tissue lysates.
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Figure 5: SENP3 upregulates the transcription of N-cadherin through de-SUMOlyation of FOXC2(A) Luciferase reporter assay in HEK293T cells co-transfected with the vector/N-cadherin promoter reporter and different doses of FOXC2. Relative luciferase activity (RLA) was shown as mean±SEM of three independent experiments. (B) The constructs of a series of N-cadherin promoter reporter with various truncations (left) and luciferase reporter assay (right) in HEK293T cells that were co-transfected with the vector/the reporter truncates and FOXC2. (C) Flag ChIP assay using the antibody against Flag and the primer of the N-cadherin promoter region −428/−209 in HEK293T cells that were transfected with the Flag-tagged vector (F-vector) / Flag-tagged wild-type FOXC2 (F-FOXC2). (D, E) Luciferase reporter assay (left) and Flag ChIP (right) in HEK293T cells that were transfected with F-FOXC2 /SUMO-less mutant of FOXC2 (F-FOXC2K214R) (D), or together with wild-type/dominant negative mutant of SENP3 (E). Both assays were repeated three times and the results were shown as mean±SEM. **: P < 0.01; ***: P<0.001. (F) The N-cadherin protein levels in the stable cell lines SGC7901-MOCK and SGC7901-SENP3 that were transiently transfected with FOXC2. (G) The SENP3 and N-cadherin protein levels in the representative gastric cancer tissue lysates.
Mentions: Although FOXC2 serves as an EMT-inducing TF, its target genes for mesenchymal induction remain unknown. We noticed that the expressions of fibronectin and N-cadherin were consistently regulated by SENP3 in various gain- or loss-of-function settings. To clarify the functions of FOXC2 in transcriptional regulation of these two genes, we constructed the luciferase reporters fused to the promoter regions of the human fibronectin and N-cadherin genes. The relative luciferase activity (RLA) of the reporter for N-cadherin was increased in a FOXC2 dose-dependent manner (Fig. 5A), indicating that FOXC2 might be the transcription factor of N-cadherin, and that the reporter, namely N-cad luc reporter, was usable. In contrast, the RLA of the reporter for fibronectin was not changed by FOXC2 overexpression (data not showed), suggesting that fibronectin was not regulated by FOXC2, despite that it could be upregulated by SENP3. N-cadherin was then selected as a representative EMT marker gene potentially regulated by SENP3 through FOXC2. To identify the FOXC2-binding elements, we measured the reporter RLA in the presence of FOXC2 based on a series of truncates of this N-cad luc reporter. The results suggested that the sequence between −428 and −209 bp on the N-cadherin promoter contained the essential elements regulated by FOXC2 (Fig. 5B). A ChIP assay was further performed, and the results verified the binding of FOXC2 at the −428 ~ −209 region (Fig. 5C).

Bottom Line: The impact of cellular oxidative stress in promoting the epithelial-mesenchymal transition (EMT) has been noticed.Meanwhile N-cadherin is verified as a target gene of FOXC2, which is transcriptionally activated by a SUMO-less FOXC2.Additionally, reactive oxygen species-induced de-SUMOylation of FOXC2 can be blocked by silencing endogenous SENP3.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Cell Biology, Shanghai Key Laboratory of Tumor Microenvironment and Inflammation, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China. Institute of Neuroscience, Wenzhou Medical University, School of Medicine, Zhejiang, China. These authors contribute equally to the work.

ABSTRACT
The impact of cellular oxidative stress in promoting the epithelial-mesenchymal transition (EMT) has been noticed. Our previous study shows that SENP3, a redox-sensitive SUMO2/3-specific protease, accumulates in a variety of cancers, but whether SENP3 and SUMOylation involve in the regulation of EMT is unclear. The present study uncovers a novel role of SENP3 in promoting the EMT process in gastric cancer via regulating an EMT-inducing transcription factor, forkhead box C2 (FOXC2). We demonstrate that the expression of mesenchymal marker genes and cell migration ability are enhanced in SENP3-overexpressing gastric cancer cells and attenuated in SENP3-knockdown cells. A nude mouse model and a set of patient's specimens suggest the correlation between SENP3 and gastric cancer metastasis. Biochemical assays identify FOXC2 as a substrate of SENP3. Meanwhile N-cadherin is verified as a target gene of FOXC2, which is transcriptionally activated by a SUMO-less FOXC2. Additionally, reactive oxygen species-induced de-SUMOylation of FOXC2 can be blocked by silencing endogenous SENP3. In conclusion, SENP3, which is increased in gastric cancer cells, potentiates the transcriptional activity of FOXC2 through de-SUMOylation, in favor of the induction of specific mesenchymal gene expression in gastric cancer metastasis.

Show MeSH
Related in: MedlinePlus